Mechanisms regulating differential activation of membrane-mediated signaling by 1alpha,25(OH)2D3 and 24R,25(OH)2D3

Vitamin D metabolites 1alpha,25(OH)(2)D(3) and 24R,25(OH)(2)D(3) regulate endochondral ossification in a cell maturation-dependent manner via membrane-mediated mechanisms. 24R,25(OH)(2)D(3) stimulates PKC activity in chondrocytes from the growth plate resting zone, whereas 1alpha,25(OH)(2)D(3) stimulates PKC in growth zone chondrocytes. We used the rat costochondral growth plate cartilage cell model to study how these responses are differentially regulated. 1alpha,25(OH)(2)D(3) acts on PKC, MAP kinase, and downstream physiological responses via phosphatidylinositol-specific PLC-beta; 24R,25(OH)(2)D(3) acts via PLD. In both cases, diacylglycerol (DAG) is increased, activating PKC. Both cell types possess membrane and nuclear receptors for 1alpha,25(OH)(2)D(3), but the mechanisms that render the 1alpha,25(OH)(2)D(3) pathway silent in resting zone cells or the 24R,25(OH)(2)D(3) pathway silent in growth zone cells are unclear. PLA(2) is pivotal in this process. 1alpha,25(OH)(2)D(3) stimulates PLA(2) activity in growth zone cells and 24R,25(OH)(2)D(3) inhibits PLA(2) activity in resting zone cells. Both processes result in PKC activation. To understand how negative regulation of PLA(2) results in increased PKC activity in resting zone cells, we used PLA(2) activating peptide to stimulate PLA(2) activity and examined cell response. PLAP is not expressed in resting zone cells in vivo, supporting the hypothesis that PLA(2) activation is inhibitory to 24R,25(OH)(2)D(3) action in these cells.     

Plasma membrane requirements for 1alpha,25(OH)2D3 dependent PKC signaling in chondrocytes and osteoblasts

1,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A(2) (PLA(2)) activity. Cells that are responsive to 1alpha,25(OH)(2)D(3) express PLA(2) activating protein (PLAA), suggesting a link between ERp60 and PLA(2). Increased PLA(2) results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCbeta), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA(2), PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1alpha,25(OH)(2)D(3). Here, we use the VDR(-/-) mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1alpha,25(OH)(2)D(3)-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1alpha,25(OH)(2)D(3). The results show that ERp60 and PLAA are localized to 1alpha,25(OH)(2)D(3)-responsive growth zone cells and metaphyseal osteoblasts, even in VDR(-/-) mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1alpha,25(OH)(2)D(3) and reduces the ability of 1alpha,25(OH)(2)D(3) to regulate [(35)S]-sulfate incorporation.     

1alpha,25(OH)2 vitamin D3 actions on ion channels in osteoblasts

Membrane-initiated cellular responses to steroids include modulation of ion channel activities via signal transduction pathways. However, the molecular mechanisms involved in nongenomic actions remain only partially understood. Our research has focused on the rapid effects of 1alpha,25(OH)(2) Vitamin D(3) [1,25D] on L-type Ca(2+) [L-Ca] and DIDS-sensitive Cl(-) channels in osteoblasts. Physiological nanomolar concentrations of hormonally active 1,25D promote rapid (1-5 min) potentiation of outward Cl(-) currents in osteosarcoma ROS 17/2.8 cells and mouse primary osteoblasts. In addition, 1,25D increases inward barium currents through L-Ca channels at low depolarizing potentials within seconds in a fashion similar to the 1,4-dihydropyridine [DHP] agonist Bay K8644. We found that second messenger cAMP is involved in 1,25D potentiation of Cl(-) and Ca(2+) channels. Nongenomic 1,25D effects on ion channel activities in osteoblasts appear to involve different mechanisms that include a possible direct interaction with the L-Ca channel molecule, on one hand, and signaling through the cAMP pathway, on the other. Rapid 1,25D actions on Cl(-) and Ca(2+) currents seem to couple to secretory activities in osteoblasts, thus contributing to bone mass formation.     

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.     

Bioactive analogs that simulate subsets of biological activities of 1alpha,25(OH)(2)D(3) in osteoblasts

1alpha,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] treatment of osteoblastic cells elicits a series of measurable responses that include both rapid, membrane-initiated effects and longer-term nuclear receptor-mediated effects. Structural analogs have been identified and characterized that selectively activate subsets of these pathways. Two analogs from over 35 that have been tested were chosen for this comparison because they activate non-overlapping response pathways, presumably representing either membrane-initiated or nuclear receptor-initiated activities. Compound AT [25(OH)-16ene-23yne-D(3)] lacks the 1-hydroxyl essential for interacting with the nuclear receptor, but triggers Ca(2+) influx through plasma membrane Ca(2+) channels, augments parathyroid hormone (PTH)-induced Ca(2+) signals, dephosphorylates the matrix protein osteopontin (OPN), and along with PTH stimulates release of calcium from calvaria in organ culture. Compound BT [1alpha,24(OH)(2)-22ene-24cyclopropyl-D(3)] does not elicit any of the rapid responses or enhance PTH-induced bone resorption, but binds to the nuclear receptor for 1alpha,25(OH)(2)D(3) and increases steady state mRNA levels of both OPN and osteocalcin over a 48 h period. Together, these two analogs recapitulate all of the known actions of 1alpha,25(OH)(2)D(3) on osteoblasts. Based on these findings, we conclude that Ca(2+) release from bone stimulated by 1alpha,25(OH)(2)D(3) and PTH is related to the rapid, membrane-initiated actions and is not likely to involve binding to the nuclear receptor for 1alpha,25(OH)(2)D(3). Longer term stimulation of bone formation by 1alpha,25(OH)(2)D(3), however, appears to involve solely the nuclear receptor-mediated effects. These findings support our model of 1alpha,25(OH)(2)D(3) as a coupling factor for bone resorption and formation during bone remodeling.     

Nongenomic regulation of protein kinase C isoforms by the vitamin D metabolites 1α,25-(OH)2D3 and 24R,25-(OH)2D3

Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation-dependent. In the present study, we examined the membrane distribution of PKC and whether 1α,25-(OH)₂D₃ and 24R,25-(OH)₂D₃ can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin-1 and inhibited by a PKC-specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform-specific anti-PKC antibodies suggested that PKCα is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKCζ is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKCζ-specific pseudosubstrate inhibitor peptide. The presence of PKCζ in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium-independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D-sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D-sensitive enzyme in plasma membranes was calcium-dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane-specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25-(OH)₂D₃ and 24,25-(OH)₂D₃ may be mediated via PKC. Further, PKCζ may be important in nongenomic, autocrine signal transduction at sites distal from the cell. © 1996 Wiley-Liss, Inc.     

Evidence that annexin II is not a putative membrane receptor for 1alpha,25(OH)2-vitamin D3

The seco-steroid hormone 1alpha,25(OH)(2)-vitamin D(3) (1,25-D(3)) is known to generate biological responses via both genomic and non-genomic rapid signal transduction pathways. The calcium regulated annexin II/p11 heterotetramer (AII(2)/p11(2)] was proposed by Baran and co-authors to be the membrane receptor responsible for mediating non-genomic, rapid actions of 1,25-D(3), based on ligand affinity labeling, competition, and saturation analysis experiments. Given the cytosolic presence of both the monomeric and heterotetrameric form of AII and their functional regulation by intracellular calcium concentrations, which are known to be affected by 1,25-D(3) rapid, non-genomic activities, we investigated in vitro the affinity of [(3)H]1,25-D(3) for the AII monomer and AII(2)/p11(2) in the absence and presence of calcium using saturation analysis and gel-filtration chromatography. Using two different techniques for separating bound from free ligand (perchlorate and hydroxylapatite (HAP)) over a series of 30 experiments, no evidence for specific binding of [(3)H]1,25-D(3) was obtained with or without the presence of 700 nM exogenous calcium, using either the AII monomer or AII(2)/p11(2). However saturable binding of [(3)H]1,25-D(3) to the lipid raft/caveolae enriched rat intestinal fraction was consistently observed (K(d) = 3.0 nM; B(max) = 45 fmols/mg total protein). AII was detected in lipid raft/caveolae enriched fractions from rat and mouse intestine and ROS 17/2.8 and NB4 cells by Western blot, but incubation in the presence of exogenous calcium did not ablate 1,25-D(3) binding as reported by Baran et al. Our results suggest that AII does not bind 1,25-D(3) in a physiologically relevant manner; however, recent studies linking AII(2)/p11(2) phosphorylation to vesicle fusion and its calcium regulated localization may make AII a possible down-stream substrate for 1,25-D(3) induced rapid cellular effects.     

Beta-1 integrins mediate substrate dependent effects of 1alpha,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. beta(1) integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1alpha,25(OH)(2)D(3) in a surface-dependent manner. To determine if beta(1) has a role in mediating osteoblast response, we silenced beta(1) expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human beta(1) to block ligand binding. beta(1)-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta(1), prostaglandin E(2), and osteoprotegerin in comparison with control cells. Moreover, beta(1)-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)(2)D(3). Anti beta(1) antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1alpha,25(OH)(2)D(3) on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta(1) plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)(2)D(3). The results also show that beta(1) mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)(2)D(3).     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

The unique action for bone metabolism of 1 alpha,25-(OH)2D3-26,23-lactone

[23 (S), 25 (R)]-1 alpha,25-Dihydroxyvitamin D3-26,23-lactone [( 23 (S),25 (R)]-1 alpha,25-(OH) 2D3-26,23-lactone) increased dose-dependently alkaline phosphatase activity in osteoblastic cells, clone MC3T3-E1, in medium containing 0.1% bovine serum albumin. The maximal stimulated enzyme activity per mg protein was 1.6-fold over that of control cultures at 250 pg/ml. The metabolite also increased collagen synthesis in a dose-related fashion. On the other hand, [23 (S),25 (R)]-1 alpha,25-(OH)2D3-26,23-lactone decreased slightly but significantly 45Ca mobilization, and blocked the resorptive action of 1 alpha,25-dihydroxyvitamin D3 but not that of parathyroid hormone, in mouse calvaria in organ culture. These results indicate that [23 (S),25 (R)]-1 alpha, 25-(OH)2D3-26,23-lactone stimulates the differentiation of osteoblasts and inhibits bone resorption in vitro.     

24R,25-(OH)(2)D(3) mediates its membrane receptor-dependent effects on protein kinase C and alkaline phosphatase via phospholipase A(2) and cyclooxygenase-1 but not cyclooxygenase-2 in growth plate chondrocytes

Recent studies have shown that 24R,25-(OH)(2)D(3) mediates its effects on growth plate chondrocytes via membrane receptors. This study examined the roles of phospholipase A(2) (PLA(2)) and cyclooxygenase (Cox) in the mechanism of action of 24R, 25-(OH)(2)D(3) in resting zone chondrocytes in order to determine whether the activity of one or both enzymes provides a regulatory checkpoint in the signaling pathway resulting in increased protein kinase C (PKC) activity. We also determined whether constitutive or inducible Cox is involved. Cultures were incubated with 24R, 25-(OH)(2)D(3) for 90 min to measure PKC or for 24 h to measure physiological responses ([(3)H]-thymidine incorporation, alkaline phosphatase-specific activity, [(35)S]-sulfate incorporation). Based on RT-PCR and Northern blot analysis, resting zone chondrocytes express mRNAs for both Cox-1 and Cox-2. Levels of mRNA for both proteins were unchanged from control levels after a 24-h incubation with 24R,25-(OH)(2)D(3). To examine the role of Cox, the cultures were also treated with resveratrol (a specific inhibitor of Cox-1), NS-398 (a specific inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). Cox-1 inhibition resulted in effects on proliferation, differentiation, and matrix production typical of 24R, 25-(OH)(2)D(3). In contrast, inhibition of Cox-2 had no effect, indicating that 24R,25-(OH)(2)D(3) exerts its effects via Cox-1. Inhibition of Cox-1 also blocked 24R,25-(OH)(2)D(3)-dependent increases in PKC. Activation of PLA(2) with melittin inhibited 24R, 25-(OH)(2)D(3)-dependent stimulation of PKC, and inhibition of PLA(2) with quinacrine stimulated PKC in response to 24R, 25-(OH)(2)D(3). Inclusion of resveratrol reduced the melittin-dependent inhibition of PLA(2) and caused an increase in quinacrine-stimulated PLA(2) activity. Metabolism of arachidonic acid to leukotrienes is not involved in the response to 24R, 25-(OH)(2)D(3) because inhibition of lipoxygenase had no effect. The effect of 24R,25-(OH)(2)D(3) was specific because 24S,25-(OH)(2)D(3), the biologically inactive stereoisomer, failed to elicit a response from the cells. These results support the hypothesis that 24R, 25-(OH)(2)D(3) exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA(2). PKC regulation may occur at multiple stages in the signal transduction cascade.     

Evidence for the metabolism of 24R-hydroxy-25-fluorovitamin D3 and 1alpha-hydroxy-25-fluorovitamin D3 to 1alpha,24R-dihydroxy-25-fluorovitamin D3.

High-pressure liquid chromatography capable of resolving all known vitamin D metabolites and a sensitive competitive binding protein assay specific for 1α,25-dihydroxyvitamin D₃ were used to assay the blood of rats dosed with ethanol, 1α-hydroxyvitamin D₃, 24R-hydroxy-25-fluorovitamin D₃, or 1α-hydroxy-25-fluorovitamin D₃. Compared to the ethanoldosed animals, the blood of rats dosed with 1α-hydroxyvitamin D₃ had increased levels of 1α,25-dihydroxyvitamin D₃; but those dosed with the fluorinated vitamins did not. Instead, their blood contained a compound that cochromatographs with 1α,24R-dihydroxyvitamin D₃ on high-pressure liquid chromatography and binds to the 1,25-dihydroxyvitamin D₃ receptor proteins. 1α,24R-Dihydroxyvitamin D₃ binds as well as 1α, 25-dihydroxyvitamin D₃ to the chick-intestinal cytosol receptor protein for 1α,25-dihydroxyvitamin D₃; whereas 1α,24S-dihydroxyvitamin D₃ binds only one-tenth as well as 1α,25-dihydroxyvitamin D₃. Thus it appears that in vivo, the fluorinated vitamin D compounds are converted to a compound likely to be 1α,24R-dihydroxy-25-fluorovitamin D₃ and that may rival the potency of 1α,25-dihydroxyvitamin D₃.     

Effects of 24R,25- and 1alpha,25-dihydroxyvitamin D3 on mineralizing growth plate chondrocytes

Time- and dosage-dependent effects of 1,25(OH)(2)D(3) and 24,25(OH)(2)D(3) on primary cultures of pre- and post-confluent avian growth plate (GP) chondrocytes were examined. Cultures were grown in either a serum-containing culture medium designed to closely mimic normal GP extracellular fluid (DATP5) or a commercially available serum-free media (HL-1) frequently used for studying skeletal cells. Hoechst DNA, Lowry protein, proteoglycan (PG), lactate dehydrogenase (LDH), and alkaline phosphatase (ALP) activity and calcium and phosphate mineral deposition in the extracellular matrix were measured. In preconfluent cultures grown in DATP5, physiological levels of 24,25(OH)(2)D(3) (0.10-10 nM) increased DNA, protein, and LDH activity significantly more than did 1,25(OH)(2)D(3) (0.01-1.0 nM). However, in HL-1, the reverse was true. Determining ratios of LDH and PG to DNA, protein, and each other, revealed that 1,25(OH)(2)D(3) specifically increased PG, whereas 24,25(OH)(2)D(3) increased LDH. Post-confluent cells were generally less responsive, especially to 24,25(OH)(2)D(3). The positive anabolic effects of 24,25(OH)(2)D(3) required serum-containing GP-fluid-like culture medium. In contrast, effects of 1,25(OH)(2)D(3) were most apparent in serum-free medium, but were still significant in serum-containing media. Administered to preconfluent cells in DATP5, 1,25(OH)(2)D(3) caused rapid, powerful, dosage-dependent inhibition of Ca(2+) and Pi deposition. The lowest level tested (0.01 nM) caused >70% inhibition during the initial stages of mineral deposition; higher levels of 1,25(OH)(2)D(3) caused progressively more profound and persistent reductions. In contrast, 24,25(OH)(2)D(3) increased mineral deposition 20-50%; it required >1 week, but the effects were specific, persistent, and largely dosage-independent. From a physiological perspective, these effects can be explained as follows: 1,25(OH)(2)D(3) levels rise in hypocalcemia; it stimulates gut absorption and releases Ca(2+) from bone to correct this deficiency. We now show that 1,25(OH)(2)D(3) also conserves Ca(2+) by inhibiting mineralization. The slow anabolic effects of 24,25(OH)(2)D(3)are consistent with its production under eucalcemic conditions which enable bone formation. These findings, which implicate serum-binding proteins and accumulation of PG in modulating accessibility of the metabolites to GP chondrocytes, also help explain some discrepancies previously reported in the literature.     

Modulation of 1alpha,25-dihydroxyvitamin D3-membrane associated, rapid response steroid binding protein expression in mouse odontoblasts by 1alpha,25-(OH)2D3

The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells.     

Isolation and identification of 1 alpha,25-dihydroxy-24-oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3: in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3

Three new in vivo metabolites of 1 alpha,25-dihydroxyvitamin D3 were isolated from the serum of dogs given large doses (two doses of 1.5 mg/dog) of 1 alpha,25-dihydroxyvitamin D3. The metabolites were isolated and purified by methanol-chloroform extraction and a series of chromatographic procedures. By cochromatography on a high-performance liquid chromatograph, ultraviolet absorption spectrophotometry, mass spectrometry, Fourier-transform infrared spectrophotometry, and specific chemical reactions, the metabolites were identified as 1 alpha,25-dihydroxy-24- oxovitamin D3, 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, and 1 alpha,24(S),25-trihydroxyvitamin D3. According to these procedures, the total amounts of the isolated metabolites were as follows: 1 alpha,25-dihydroxyvitamin D3, 23.6 micrograms; 1 alpha,25-dihydroxy-24- oxovitamin D3, 1.8 micrograms; 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 9.2 micrograms; 1 alpha,24(R),25-trihydroxyvitamin D3, 15.4 micrograms; 1 alpha,24(S),25-trihydroxyvitamin D3, 1.0 microgram. With recovery corrections, the serum levels of each metabolite were approximately 49 ng/mL for 1 alpha,25-dihydroxyvitamin D3, 3.7 ng/mL for 1 alpha,25-dihydroxy-24- oxovitamin D3, 19 ng/mL for 1 alpha,25-dihydroxyvitamin D3 26,23-lactone, 32 ng/mL for 1 alpha,24(R),25-trihydroxyvitamin D3, and 2.1 ng/mL for 1 alpha,24(S),25-trihydroxyvitamin D3.     

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.     

Novel vitamin D3 antipsoriatic antedrugs: 16-En-22-oxa-1alpha,25-(OH)2D3 analogs

A series of 16-en-22-oxa-derivatives of vitamin D3 based on the structure of maxacalcitol (2) were prepared. Maxacalcitol is currently used topically for the treatment of psoriasis and is recognized as the most successful antedrug of natural vitamin D(3) because it retains the original antiproliferative activity of calcitriol without increased calcemic activity. We introduced 16-olefinic functionality to accelerate the oxidative metabolism of the drug in liver, presumed to be essential for the reduction of calcemic activity, and modified the side-chain moiety by placing the 22-oxygen on the more labile allylic carbon center. Novel 22-oxa analogs (7a-i), carrying either the 24-alkynyl bond or 24-hydroxy functionality in addition to the 16-double bond were synthesized and their pharmacokinetics were evaluated.